Microchimica Acta

, 186:123 | Cite as

Colorimetric determination of ascorbic acid and the activity of alkaline phosphatase based on the inhibition of the peroxidase-like activity of citric acid-capped Prussian Blue nanocubes

  • Tengteng Wu
  • Wenli Hou
  • Zhangyan Ma
  • Meiling LiuEmail author
  • Xiaoying LiuEmail author
  • Youyu Zhang
  • Shouzhuo Yao
Original Paper


Colorimetric methods are described for the determination of ascorbic acid (AA) and alkaline phosphatase (ALP). Both assays are based on the inhibition of the peroxidase (POx)-like activity of Prussian Blue nanocubes (PB NCs) capped with citric acid. They catalyze the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to produce a blue color with an absorption maximum at 652 nm. On addition of AA, the PB NCs are reduced to Prussian White (PW) which does not act as a POx mimic. This results in a decreased rate of the formation of the blue coloration whose intensity decreases with increasing concentration of AA. The assay allows AA to be quantified with a 35 nM detection limit (at 3σ/m). The hydrolysis of AA phosphate by ALP leads to the formation of AA which can be quantified by the above method. Based on this, the activity of ALP can be determined by measurement of the intensity of the blue coloration thus formed. The method can be used to determine the activity of ALP with a detection limit as low as 0.23 U·L−1.

Graphical abstract

Schematic presentation of a method for colorimetric determination of ALP activity. AA obtained by ALP-catalyzed hydrolysis of ascorbic acid phosphate (AAP) inhibits the intrinsic peroxidase-like activity of PB NCs by reducing Prussian Blue nanocrystals (PB NCs) to form inactive Prussian White (PW).


Enzyme mimic Nanocubes Prussian White Peroxidase mimetic 3,3,5,5-Tetramethylbenzidine Inhibition Colorimetric assay 



This work was supported by the National Natural Science Foundation of China (21645008, 21305042, 21375037), Scientific Research Fund of Hunan Provincial Education Department (14B116), Science and Technology Department (14JJ4030), the construct program of the key discipline in Hunan province and the Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province.

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2018_3224_MOESM1_ESM.docx (3.4 mb)
ESM 1 (DOCX 3.36 mb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical EngineeringHunan Normal UniversityChangshaPeople’s Republic of China
  2. 2.College of ScienceHunan Agricultural UniversityChangshaPeople’s Republic of China

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